Ⅰ. INTRODUCTION
Alfalfa (Medicago sativa L.) is a perennial forage legume widely known as the “Queen of Forages.” It is extensively utilized as a high-quality feed for ruminant livestock due to its high biomass productivity and crude protein (CP) concentration (Park et al., 2005;McDonald et al., 2021). In addition, alfalfa provides substantial ecological and agricultural value through its ability to fix atmospheric nitrogen via symbiosis with rhizobia, thereby contributing to the nitrogen supply in forage production systems (Park et al., 2005). However, alfalfa is highly sensitive to environmental and edaphic conditions such as various productivity-limiting factors, including soil pH, drainage, nutrient availability, and rhizobial activity, can significantly compromise its yield and persistence (Undersander et al., 2011).
Among the critical nutrients affecting alfalfa, boron (B) is an essential micronutrient involved in cell wall formation, meristematic development, leaf retention, and regrowth. Conversely, B deficiency can impair growing point development and cause leaf chlorosis, defoliation, and delayed regrowth, ultimately reducing dry matter yield(DMY) and forage nutritive value (Wright, 1986). Because alfalfa has a relatively high requirement for B, boron fertilization is considered a vital management practice for successful stand establishment and production (Undersander et al., 2011). Molybdenum (Mo) is another important micronutrient associated with nitrogen fixation and metabolism, playing a key role in nodulation and symbiotic nitrogen fixation (Meyer et al., 1997). When Mo is deficient, nitrogen fixation efficiency decreases, which limits nitrogen uptake, vegetative growth, DMY, and CP concentration.
Rhizobial inoculation is also a key biological factor determining the nitrogen-supplying capacity of alfalfa. If rhizobia are not adequately established in the soil, biological nitrogen fixation is severely limited, resulting in suppressed growth and productivity (Han et al., 2024). Agricultural lime application is commonly used to correct soil acidity, improve calcium (Ca) and magnesium (Mg) availability, and create favorable conditions for rhizobial activity. Alfalfa is highly sensitive to soil acidity and thrives best under near-neutral pH conditions. Previous studies have reported that both nodulation and nitrogen fixation decline when soil pH drops below 6.5, leading to reduced productivity (Hannaway et al., 2019). Therefore, the absence of lime application represents a major potential limiting factor affecting soil pH, Ca supply, and rhizobial efficacy.
Previous studies on alfalfa productivity-limiting factors have often focused on acidic soils, where responses to liming, Mo application, and rhizobial activity are expected to be more pronounced. Under such conditions, the negative effects of low pH on root growth, nodulation, nutrient availability, and biological nitrogen fixation are relatively clear. However, it remains uncertain whether these factors still limit alfalfa productivity when the initial soil pH is approximately 6.5, which is close to the lower boundary of the suitable pH range for alfalfa production. This question is important because productivity reduction under such soil conditions would indicate that alfalfa yield is not controlled only by severe soil acidity, but also by the combined effects of micronutrient availability, rhizobial establishment, Ca supply, and pH-related soil amendment practices.
Although B and Mo are micronutrients, rhizobia serve as biological agents, and lime acts as a soil amendment, all of these components interact to influence the early growth, nitrogen fixation, regrowth, and DMY of alfalfa. Therefore, evaluating their relative effects under identical experimental conditions is crucial for identifying the primary constraints in alfalfa production. While these factors are individually known to affect alfalfa performance, their relative impacts have yet to be comprehensively quantified.
Therefore, this study was conducted to evaluate the effects of B limitation, Mo limitation, lack of rhizobial inoculation, and lime limitation on DMY and forage nutritive value of alfalfa grown under soil conditions with an initial pH of approximately 6.5. The main objective was to identify the primary productivity-limiting factors affecting alfalfa production under relatively favorable soil pH conditions.
Ⅱ. MATERIALS AND METHODS
This experiment was conducted in a greenhouse at the Department of Animal Resources Development, National Institute of Animal Science, Cheonan, Republic of Korea. Alfalfa was sown on April 8, 2022, and the experiment continued until August 2, 2024. The alfalfa cultivar used in this study was ‘SW5615’ with a fall dormancy rating 5. Seeds were sown at a rate equivalent to 20 kg ha−1 in experimental plots measuring 0.5 m × 0.6 m (0.30 m2).
The soil used in this experiment was collected from the experimental field of the National Institute of Animal Science. The initial soil pH was approximately 6.5. Basal fertilizer was applied at rates of 100, 300, and 300 kg ha−1 for nitrogen (N), phosphorus (P), and potassium (K), respectively. Nitrogen was applied entirely as a basal fertilizer at sowing, while P and K were split-applied according to each harvest period.
The experiment was arranged in a randomized complete block design with three replications. Treatments were established based on productivity-limiting factors and consisted of five treatments: control, B limitation, Mo limitation, rhizobium limitation, and lime limitation. In the control treatment, B, Mo, rhizobial inoculant, and lime were all supplied. In each limitation treatment, one of these factors was omitted: B was not applied in the B limitation treatment, Mo was not applied in the Mo limitation treatment, rhizobial inoculant was not applied in the rhizobium limitation treatment, and lime was not applied in the lime limitation treatment. Soil moisture was maintained to avoid excessive water deficit, while temperature and light conditions were not artificially controlled, following natural greenhouse conditions.
Alfalfa was harvested at the early flowering stage, corresponding to approximately 10% bloom. A total of five harvests were conducted during the experimental period. Harvested herbage was dried at 65°C for 72 h to determine dry matter (DM). DMY was calculated by multiplying fresh matter yield by DM. Total DMY was calculated as the sum of the five harvests. Dried samples were ground to pass through a 1-mm screen and stored in a cool and shaded place until forage quality analysis.
The measured parameters included DMY and forage quality included chemical composition and nutritive value. Chemical composition included CP, neutral detergent fiber (NDF), and acid detergent fiber (ADF), whereas forage nutritive value was assessed using total digestible nutrients (TDN) and relative feed value (RFV). Forage quality analysis was conducted according to the methods of the Association of Official Analytical Chemists (AOAC, 1990). CP concentration was determined using the Dumas combustion method (Chang and Zhang, 2017) with an elemental analyzer (Vario MAX CUBE, Elementar, Germany). NDF and ADF were analyzed using an Ankom200 fiber analyzer (Ankom Technology Corp., Macedon, NY, USA) according to the method of Goering and Van Soest (1970). TDN was calculated using the equation , as described by Holland et al. (1990). RFV was calculated using the equation , as described by Jeranyama and Garcia (2004).
Statistical analysis was performed using R software version 4.4.2 (R Core Team, 2024). Analysis of variance (ANOVA) was conducted using the stats and agricolae packages to test treatment effects at a significance level of 0.05. When significant differences were detected, treatment means were compared using the least significant difference (LSD) test.
Ⅲ. RESULTS AND DISCUSSION
1. Dry matter yield
Productivity-limiting factor treatments significantly affected alfalfa DMY (p<0.05; Fig. 1). The control treatment, in which all factors were supplied, produced 31,940 kg ha−1 of DMY. The DMY values of the B limitation, Mo limitation, rhizobium limitation, and lime limitation treatments were 31,244, 26,258, 20,150, and 20,628 kg ha−1, respectively. Compared with the control, the relative DMY of the B limitation treatment was 97.8%, showing no significant difference from the control. In contrast, the DMY values of the Mo limitation, rhizobium limitation, and lime limitation treatments decreased to 82.2, 63.1, and 64.6% of the control, respectively. In particular, the greatest reductions in DMY were observed under rhizobium and lime limitation treatments, indicating that alfalfa productivity was strongly affected by rhizobial inoculation and lime application.
B is an essential micronutrient for alfalfa growth and is involved in cell wall formation, growing point development, and leaf retention. However, in the present study, the DMY of the B limitation treatment was not significantly different from that of the control (p>0.05). Kheirkhah et al. (2016) reported that B application had a positive effect on alfalfa production. In contrast, Sapkota et al. (2018) reported that B fertilization affected DMY in some individual harvests, but it did not significantly affect annual DMY. This result is consistent with the present study, suggesting that the effect of B limitation on alfalfa yield may depend on soil B status, plant B concentration, and growing conditions.
Mo limitation treatment showed significantly lower DMY than the control (p<0.05). Molybdenum is a micronutrient involved in nitrogen fixation and nitrogen metabolism, and Mo deficiency can reduce nitrogen fixation efficiency in nodules, thereby limiting alfalfa growth (Meyer et al., 1997). Therefore, the reduction in DMY under the Mo limitation in the present study may be associated with reduced biological nitrogen fixation.
Rhizobium limitation treatment resulted in the greatest reduction in DMY (p<0.05). Alfalfa fixes atmospheric nitrogen through symbiosis with rhizobia, and this process plays an important role in plant growth and protein production (Han et al., 2024). If rhizobia are not adequately established, nitrogen supply can be limited, resulting in reduced growth and DMY. In the present study, DMY under rhizobium limitation was only 63.1% of the control, indicating that rhizobial inoculation or nodulation is a key factor for maintaining alfalfa productivity.
Lime limitation treatment also showed significantly lower DMY than the control (p<0.05). Lime application can improve soil pH, increase Ca availability, promote root growth, and enhance rhizobial activity (Moreira and Fageria, 2010). Although the initial soil pH in this study was approximately 6.5, which is close to the suitable range for alfalfa growth, the marked reduction in DMY under lime limitation suggests that lime application may have affected alfalfa productivity not only through pH correction but also through improved Ca supply, rhizosphere conditions, and rhizobial activity. Therefore, lime application may be an important management practice for stable alfalfa production.
2. Chemical composition and forage nutritive value
Productivity-limiting factor treatments significantly affected CP concentration of alfalfa (p<0.05; Fig. 2A), whereas NDF and ADF were not significantly affected (p>0.05; Fig. 2B-C). The highest CP concentration was observed in the lime limitation treatment, while there were no clear differences among the control, B limitation, Mo limitation, and rhizobium limitation treatments.
Also, forage nutritive value, such as TDN and RFV, was not significantly affected by the treatment (p>0.05; Fig. 3A-B).
Previous studies have reported that B application can increase CP concentration and improve the forage nutritive value of alfalfa (Wright, 1986;Turan et al., 2010). However, in the present study, B limitation did not significantly affect CP, NDF, ADF, TDN, or RFV. Similarly, Sapkota et al. (2018) reported that B fertilization did not significantly affect the forage nutritive value of irrigated alfalfa. These results suggest that B limitation may not alter forage nutritive value when physiological B deficiency is not severe enough to affect plant growth or forage composition. The effect of B on alfalfa nutritive value may also be more dependent on soil B availability, plant B status, and environmental conditions than on B treatment alone. Mo and rhizobium limitation were expected to reduce CP concentration because both factors are closely associated with biological nitrogen fixation. However, CP concentration was not significantly reduced by Mo or rhizobium limitation in this study (p>0.05). This suggests that the effect of reduced nitrogen fixation was reflected more strongly in DMY reduction than in CP concentration. Because CP concentration is expressed on a concentration basis, it may not fully represent total protein productivity when DMY differs greatly among treatments.
The significantly higher CP concentration observed under lime limitation should not be interpreted as an improvement in forage quality caused by lime limitation. Rather, it was likely associated with reduced biomass accumulation or delayed maturity (Rocateli and Zhang, 2015). In general, as alfalfa matures, stem proportion and fiber concentration increase, whereas CP concentration decreases. Because the lime limitation treatment showed markedly lower DMY than the control in the present study, the higher CP concentration under lime limitation may have resulted from slower growth, delayed maturity, or a higher proportion of relatively young tissues. Therefore, the increase in CP concentration under lime limitation should be interpreted as a concentration effect associated with growth suppression rather than an actual increase in protein productivity.
Overall, this study demonstrated that alfalfa productivity was more strongly affected by DMY reduction than by changes in forage nutritive value by productivity-limiting factors under soil conditions with an initial pH of approximately 6.5. Lack of rhizobial inoculation and lime limitation were identified as the major productivity-limiting factors, followed by Mo limitation, whereas B limitation did not significantly reduce DMY or forage nutritive value. These results suggest that biological nitrogen fixation and lime-related soil conditions remain critical for alfalfa production even when soil pH is close to the suitable range for alfalfa growth. The increase in CP concentration under lime limitation was likely caused by reduced biomass accumulation or delayed maturity rather than improved forage quality. Therefore, rhizobial inoculation, lime application, and Mo management should be considered important practices for maintaining stable alfalfa productivity under relatively favorable soil pH conditions.
